Evidence Supporting That C-to-U RNA Editing Is the Major Force That Drives SARS-CoV-2 Evolution

Abstract

Mutations of DNA organisms are introduced by replication errors. However, SARS-CoV-2, as an RNA virus, is additionally subjected to rampant RNA editing by hosts. Both resources contributed to SARS-CoV-2 mutation and evolution, but the relative prevalence of the two origins is unknown. We performed comparative genomic analyses at intra-species (world-wide SARS-CoV-2 strains) and inter-species (SARS-CoV-2 and RaTG13 divergence) levels. We made prior predictions of the proportion of each mutation type (nucleotide substitution) under different scenarios and compared the observed versus the expected. C-to-T alteration, representing C-to-U editing, is far more abundant that all other mutation types. Derived allele frequency (DAF) as well as novel mutation rate of C-to-T are the highest in SARS-CoV-2 population, and C-T substitution dominates the divergence sites between SARS-CoV-2 and RaTG13. This is compelling evidence suggesting that C-to-U RNA editing is the major source of SARS-CoV-2 mutation. While replication errors serve as a baseline of novel mutation rate, the C-to-U editing has elevated the mutation rate for orders of magnitudes and accelerates the evolution of the virus.

Keywords: Allele frequency; C-to-U RNA editing; Evolution; Mutation; SARS-CoV-2.

Fig. 1

Introduction of basic evolutionary concepts. A Sequence of outgroup species is used for inference of ancestral state of the target species. Then the direction and derived allele frequency (DAF) of polymorphic sites could be determined. B Site1 has higher novel mutation rate than site2, then site1 eventually has higher DAF. C Site1 has similar novel mutation rate with site2, but site1 is positively selected and finally has higher DAF than site2. D Synonymous mutations are (believed to be) free from natural selection. The final DAF of synonymous sites might mirror the initial mutation rate

Fig. 2

Prior prediction of mutation profile based on different mutation sources. A The three possible sources of mutations in SARS-CoV-2. RDRP-mediated replication errors, ADAR-mediated A-to-I editing, and APOBEC-mediated C-to-U editing. B If replication error is the major mutation source, then the mutation profile should be symmetric. SNP denotes “single nucleotide polymorphism”. C If A-to-I editing is the major mutation source, then the A-to-G mutations should be dominant. D If C-to-U editing is the major mutation source, then the C-to-T mutations should be dominant

Fig. 3

The numbers of SNV (single nucleotide variation) in the SARS-CoV-2 genome under different thresholds of DAF (derived allele frequency). A When DAF is low, the mutation profile resembles the SNP profile. B The fraction of C-to-T substitution increases with DAF. (C-D) When DAF is high, C-to-U editing sites become dominant. E DAF spectrum of C-to-T substitutions. F DAF spectrum of non-C-to-T mutations. G Comparison of C-to-U and non-C-to-U mutations. The difference of DAF was determined by KS test

Fig. 4

The DAF of each mutation type. A The median and mean DAF of each mutation type. B Synonymous and missense sites are shown separately. Synonymous sites have obviously higher DAF than missense sites

Fig. 5

The definition of inter-species divergence. A How mutations within a population lead to speciation and the sequence divergence. B Calculating the numbers of each substitution type when outgroup (MERS-CoV) is absent or present

Fig. 6

The divergence sites between SARS-CoV-2 and RaTG13. Synonymous and missense sites were displayed separately. Non-directional mutations were counted without outgroup information. Directional mutations were inferred based on other coronaviruses as outgroup

Fig. 7

The divergence between five entomopoxviruses (EPV). A Phylogeny of the five viral sequences. B, C, D The directional inter-species divergence between two viral sequences